2.1.6. Triterpenoids

Triterpenic compounds are common constituents of plants, occurring in the form of free acids or aglycones of triterpenoid saponins [20] offering a wide range of health-promoting activities, both as pure substances or as blends [21]. Despite their beneficial properties, there is relatively little information available on their distribution in olive leaves. To our knowledge, this is the first report on their concentration in olive leaves in response to drying and season. In this work, three triterpenic acids could be identified in the different olive leaf extracts. As demonstrated in Figure 2, peak 18 showed an ion at *m/z* 471.3480 eluting at 14.71 min which was identified as maslinic acid. Peaks that eluted at 16.30 and 16.45 min gave ions at *m/z* 455.3531 and were identified as oleanolic acid and ursolic acid, respectively (Figure 2).

### *2.2. Quantification of Phytochemicals in Olive Leaf Extracts*

The phytochemicals were quantified in the different olive leaf extracts using the analytical standard calibration curves. Figure 3 shows the variation of the determined amounts of the identified phytochemicals. For phenolic compounds, oleuropein and derivatives were quantified using the calibration curve of the external standard oleuropein. Hydroxytyrosol, ferulic acid, pinoresinol, luteolin, apigenin, diosmetin, and quercetin were quantified using their corresponding standard calibration curves. Acetoxypinoresinol and syringaresinol were quantified using the calibration curve of pinoresinol. For triterpenoids, oleanolic acid and ursolic acid were quantified with the calibration curve of oleanolic acid, whereas maslinic acid was quantified with the calibration curve of maslinic acid. Finally, benzyl alcohol pentose was quantified using the calibration curve of hydroxytyrosol.

2.2.1. Variation of Phenolic and Triterpenoid Compounds Depending on Sampling Time and Drying Temperature

Phenolic and triterpenic compounds were determined, and their amounts expressed as μg/g of olive leaf are summarized in Figure 3. Significant variation (*p* < 0.05, Supplementary Materials) was observed among samples according to the drying temperature and sampling season.

### Effect of Sampling Time

The seasonal changes in the composition of the leaves of the El Hor olive cultivar have been investigated with respect to sampling time: January, April, August, and November. The quantity and distribution of phenolic and triterpenic compounds in the leaves showed significant differences among samples of different seasons (*p* < 0.05).

As shown in Figure 4, contents in secoiridoids varied significantly among sampling time (*p* < 0.05). In fresh leaves, the highest amount was observed in leaves collected in summer and did not exceed 1 μg/g. However, for dried leaves, the highest amounts were registered for leaves collected in November (338.8 μg/g) except for leaves dried at 25 ◦C. In the latter, the January sampling time presented the highest amounts (24.5 μg/g) which decreased to 0.6 μg/g in August to slightly increase in November to reach 6.52 μg/g. As reported by other researchers, the contents of oleuropein in olive leaves were in general barely influenced by the collecting period factor [22].

It was reported that December was the sampling time during which oleuropein presented the maximum amount for 'Leccino' cultivar [4]. In another work, the samples of leaves collected in October seemed to contain slightly lower amounts of oleuropein than those collected in March, likely due to a higher degradation rate of this glycoside in autumn and in addition to lower production of green young leaves (leaf renovation) during this season with respect to the spring period [22].

The variations in lignans amounts observed in olive leaves according to sampling time were affected by the drying temperature. In fact, fresh leaves did not show the same behavior as observed in dried leaves. As shown in Figure 4, lignans in fresh leaves decreased from January (12.6 μg/g) to April (4.0 μg/g) and re-increased in August (9.1 μg/g) and decreased in November, to reach values similar to those observed in April (4.2 μg/g). However, in leaves dried at 25 and 60 ◦C, a slight increase was observed from January to April in contrast to leaves dried at 40 ◦C, where lignans amounts, after a decrease from January to April, increased in August and November to reach 2.41 μg/g. As far as we know, this is the first time the behavior of lignans has been studied according to drying.

Luteolin and quercetin were detected only in the November sampling time, whereas diosmetin was detected in samples from each period. Flavonoids amounts showed significant variations (*p* < 0.05) among samples, however, they did not exceed 3.1 μg/g according to the sampling time. The highest values were observed in August and November for fresh leaves and when leaves were dried at 120 ◦C (Figure 4).

**Figure 3.** Variation of phytochemicals identified in olive leaf extracts. (J) January, (A) April, (Ag) August, (N) November, FL: Fresh leaves. Triterpenoids are expressed as mg/g, the rest of the phytochemicals are expressed as μg/g.

**Figure 4.** Variation of phytochemical classes according to sampling time and drying. (J) January, (A) April, (Ag) August and (N) November, FL: fresh leaves.

Total triterpenoids showed a significant variation (*p* < 0.05) between olive leaf samples according to sampling time. These metabolites were almost absent in April (Figure 3). The highest value of total triterpenoids was registered for extracts of the November and August sampling times, in which it reached 18.8 and 27.4 mg/g, respectively. The amounts in January varied between 0.1 and 11.9 mg/g. Regardless of the season, generally, ursolic and oleanolic acid presented the major fraction of triterpenoids, whereas maslinic acid presented the lowest amounts.
